We have used fluorescence microscopy, fluorescence photobleaching recovery (FPR), and atomic force microscopy (AFM) to investigate the formation of tethered lipid bilayers on plane aluminum oxide or glass surfaces. The bilayers were assembled with the help of a two-step methodology recently proposed for microporous templates (Proux-Delrouyre et al. J. Am. Chem. Soc. 2001, 123, 8313). The first step consists of the accumulation of intact biotinylated vesicles (PC + DOPE) on a streptavidin sublayer itself immobilized on the substrate. The second step, clearly time separated, is the deliberate triggering of bilayer formation with the help of poly(ethylene glycol) (PEG), a fusion agent of lipidic vesicles. AFM and FPR measurements confirm that the vesicles do not spontaneously fuse during the first step provided that the streptavidin sublayer is present on the substrate. On the contrary, the treatment with PEG provokes the fast formation of a continuous lipid bilayer, as attested at the hundred nanometer scale by the AFM images and at the hundred micrometer scale by the lateral diffusion of a fluorescent probe (D = 2.2 x 10(-8) cm(2) s(-1) for NBD-DMPE at 22 degreesC).